During the menstrual cycle, hormone-driven functional and morphological changes occur in the female brain. The influence of hormonal contraceptives on these changes has received only little attention in the medical literature. The purpose of our study is to measure regional gray matter volume changes as a function of the cycle phase and use of hormonal contraceptives, in relation to blood concentrations of sex hormones. We performed a prospective study in 30 healthy young women; 15 women had a natural menstrual cycle and 15 were using monophasic combined hormonal contraceptives. MRI examinations were acquired at 2 specific time-points in the cycle (follicular and luteal phase). MRI studies included a T1-weighted, isotropic, high-resolution 3-D gradient echo acquisition, for the purpose of performing voxel based morphometry. Peripheral venous blood samples were obtained to determine concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH), estradiol, and progesterone. We found a highly significant negative correlation of regional gray matter volume in the anterior cingulate cortex with estradiol concentrations. To the best of our knowledge, this result has not been described before, and was only present in the natural cycle group, not in women using hormonal contraceptives. The anterior cingulate cortex is involved in emotion processing and there is literature describing behavioral alternations with changing hormone levels. Our findings provide a structural, morphological basis to support these data. Therefore, we advise neuroscientists to take into account the menstrual cycle phase and use of hormonal contraceptives, in order to avoid obtaining heterogeneous data sets, leading to a significant loss of accuracy and precision.